Ca2+ is involved in every aspect of cellular life. Ca2+-activated ion channels, mainly Ca2+-activated K+ (KCa) channels, Ca2+-activated Cl- channels (CaCC) and Ca2+-activated non-selective cation (CAN channels), sense changes of internal Ca2+ level and actively regulate membrane excitability, ion homeostasis and downstream cell signaling cascade. These channels are abundantly expressed and actively involved in the physiology of the circulatory and nervous systems, and have been linked to pathophysiology of stroke and many neurological disorders, such as epilepsy, pain, and movement disorders including ataxia and tremor. Compared with KCa channels, CaCCs and CANs are much less understood mainly due to the difficulties involved in their molecular identification. In 2008, our laboratory identified that TMEM16A and 16B in the TMEM16 family form the long- sought CaCCs. I recently demonstrated that TMEM16F, instead of being a CaCC, forms a novel CAN channel with small single channel conductance (SCAN) and minimal anion permeability. Our in vitro and in vivo experiments further illustrate that TMEM16F-SCAN channels are required for lipid scrambling in platelets during blood coagulation and TMEM16F knockout mice exhibit bleeding defects and protection in carotid artery thrombosis associated with platelet deficiency in Ca2+-dependent phosphatidylserine (PS) exposure. The focus of this proposal is to define the molecular properties of Ca2+-activated TMEM16 channels and their cellular functions in platelets and in cerebellar Purkinje cells. In Aim I, I will pinpoint the key residues that are important for ion selectivity and Ca2+ binding to understand the molecular mechanisms underlying the functions of TMEM16 channels. In Aim II, I will investigate the physiological functions of the TMEM16F-SCAN channels in platelets and cerebellar Purkinje cells using a combination of electrophysiology and in vitro/in vivo imaging techniques. Studies of this proposal will facilitate the understanding of TMEM16 channels at the molecular level, help define their physiological functions, and provide insights to develop new therapeutics to target these channels to prevent and treat neurological disorders and stroke.